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Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480911/ https://www.ncbi.nlm.nih.gov/pubmed/26108968 http://dx.doi.org/10.1186/s12864-015-1671-5 |
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author | Katayama, Shintaro Skoog, Tiina Jouhilahti, Eeva-Mari Siitonen, H. Annika Nuutila, Kristo Tervaniemi, Mari H Vuola, Jyrki Johnsson, Anna Lönnerberg, Peter Linnarsson, Sten Elomaa, Outi Kankuri, Esko Kere, Juha |
author_facet | Katayama, Shintaro Skoog, Tiina Jouhilahti, Eeva-Mari Siitonen, H. Annika Nuutila, Kristo Tervaniemi, Mari H Vuola, Jyrki Johnsson, Anna Lönnerberg, Peter Linnarsson, Sten Elomaa, Outi Kankuri, Esko Kere, Juha |
author_sort | Katayama, Shintaro |
collection | PubMed |
description | BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4480911 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-44809112015-06-26 Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control Katayama, Shintaro Skoog, Tiina Jouhilahti, Eeva-Mari Siitonen, H. Annika Nuutila, Kristo Tervaniemi, Mari H Vuola, Jyrki Johnsson, Anna Lönnerberg, Peter Linnarsson, Sten Elomaa, Outi Kankuri, Esko Kere, Juha BMC Genomics Research Article BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users. BioMed Central 2015-06-25 /pmc/articles/PMC4480911/ /pubmed/26108968 http://dx.doi.org/10.1186/s12864-015-1671-5 Text en © Katayama et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Katayama, Shintaro Skoog, Tiina Jouhilahti, Eeva-Mari Siitonen, H. Annika Nuutila, Kristo Tervaniemi, Mari H Vuola, Jyrki Johnsson, Anna Lönnerberg, Peter Linnarsson, Sten Elomaa, Outi Kankuri, Esko Kere, Juha Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title | Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title_full | Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title_fullStr | Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title_full_unstemmed | Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title_short | Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control |
title_sort | gene expression analysis of skin grafts and cultured keratinocytes using synthetic rna normalization reveals insights into differentiation and growth control |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480911/ https://www.ncbi.nlm.nih.gov/pubmed/26108968 http://dx.doi.org/10.1186/s12864-015-1671-5 |
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